Neuromuscular junction (“NMJ”) formation is a complex process that depends on many variables. Unfortunately, current techniques for producing NMJs suffer from one or more drawbacks which hinder their reproducibility and usefulness.
One such technique is to produce NMJs in vitro using motoneuron (“MN”)-muscle cell co-cultures. MN-muscle co-cultures have been described in Xenopus (5, 6), chick (7-9), mouse (10, 11) and rat tissues (12, 13), as well as in cross-species investigations between mouse MN-chick muscle (11, 14) and utilizing embryonic human stem cell-derived MNs-synapsed to myotubes from the C2C12 cell line (15). The drawback to these in vitro motoneuron-muscle co-culture systems is that they use serum containing media and a biological substrate (7-9, 12, 13). Since the serum containing medium contains many unknown components and because of the technical difficulties in creating reproducible biological substrates, these examples have led to undesired culture variability, making it extremely difficult, if not impossible, to ascertain the minimum factors required for recreating or maintaining the NMJ in vitro.
Only one study reports the formation of NMJs between co-cultured MNs and skeletal muscle in a defined in vitro environment. In that study, NMJs were reported to form between rat MNs and rat skeletal muscle. Numerous studies have attempted to implant human stem cells into rat models to determine if they could be of therapeutic use in disease or spinal injury models, albeit with little evidence of neuromuscular junction (NMJ) formation. No previous in vitro studies have demonstrated NMJ formation using a co-culture containing human motoneurons in a defined environment.
The inventors have previously reported a defined serum-free system for the culture of rat skeletal muscle (16), embryonic and adult rat spinal cord neurons (17, 18), and for the co-culture of rat MNs and rat embryonic skeletal muscle (19). Presently, the in vivo model of choice for investigating the therapeutic potential of stem cells in disease models such as amyotrophic lateral sclerosis (ALS) (1, 2) and in spinal cord injury (3, 4) is a system using motoneurons derived from human stem cells and rat myotubes. Accordingly, there is a need in the art for a system for NMJ formation that does not suffer from one or more of the above described drawbacks.